Promotion of Stomatal Opening by Indoleacetic Acid and Ethrel in Epidermal Strips of Vicia faba L

Indole-3-acetic acid (IAA), at concentrations of 0.01 to 1.0 millimolar, and ethephon (0.3% v/v Ethrel) promote stomatal opening when applied to epidermal peels of Vicia faba L. in light or dark. The effect of ethylene is seen by 30 minutes and maximal opening (over two times that of untreated controls) occurs after only 60 to 90 minutes in the light. Stomatal opening by IAA and Ethrel in both light and dark is prevented by 0.14 millimolar AgCl. It is suggested that the effect of added IAA, but not that of light, is linked to ethylene production. The possible role of ethylene in stomatal opening during fungal infection is discussed. The stomates of Vicia faba provide a new system to study the effects of ethylene on certain membrane-regulated processes.     

A neighboring-inhibition model for stomate patterning

The patternization of stomate distribution was investigated in the first leaf of the sporophyte of the fern Dryopteris thelypteris and in the leaves of the jade plant Crassula argentae. In the fern leaf, stomates arise over a period of 1.1 days (26.4 hr) and attain a frequency of 0.186 of the epidermal cells while in the jade plant the formative period is over only 0.8 days (19.2 hr) and stomates reach a frequency of 0.090 of the epidermal cells. A computer model was devised to simulate the appearance of stomates by an induction process for a new stomate which then inhibits contiguous cells from becoming stomates. The validity of the model was demonstrated in that it gave values for stomate and stomate cluster frequencies upon calculating the frequency of free cells. For the fern leaf, the model required 57 iteration intervals, six iterations for the time from stomate induction until the adjacent cells were inhibited (a period of induction plus inhibitions), and an induction rate of 0.02 cells per time interval. From these theoretical values and the measured period of 26.4 hr when stomates arise during leaf development, a period of induction/inhibitions is calculated as 2.7 hr. In the model for the jade plant, 81 iteration intervals are required with six iterations per period of induction/inhibitions along with an induction rate of 0.002 cells per interation interval. These values give the duration for a period of induction/inhibitions in jade of 1.4 hr. This study describes the patterning process of stomate formation by an explicit, mathematical algorithm, and, from measurements of actual leaves, the various periods in the model can be assigned real time values.     

Circadian Stomatal Rhythms in Epidermal Peels from Vicia faba

Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 30°C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

In vitro propagation of the Egyptian medicinal plant,Echinops spinosissimus Turra

The in vitro propagation of medicinal species is proving to be one successful approach to addressing the issues raised by increasing global demand, overharvest of wild populations and variability in product quality. In this report we describe a protocol for establishing nodal cultures of an Egyptian medicinal plant, Echinops spinosis-simus Turra and for the induction of regeneration in epidermal peels and leaf explants obtained from the nodal cultures. Kinetin was found to be optimal for induction and proliferation of nodal cultures. Transverse slices of leaf blades produced regenerants resembling somatic embryos in the presence of thidiazuron while regeneration was observed on epidermal peels only when the medium contained elevated auxin concentrations.     

VARIATION OF STOMATAL DISTRIBUTION IN CAREX AQUATILIS (CYPERACEAE)

The density and distribution of stomates in Carex aquatilis Wahl. in the Pacific Northwest were examined using epidermal peels of samples of leaves from natural populations, from greenhouse-grown transplants and from seedling families grown under controlled conditions. These were compared to stomatal distributions of populations in eastern North America. Populations of Carex aquatilis Wahl. form 2 groups based on the distribution and density of stomates. Carex aquatilis var. dives (Holm) Kükenthal is epistomatic, with adaxial stomatal densities of 28.7–48.5/0.1 mm². The C. aquatilis var. aquatilis is amphistomatic, with adaxial stomatal densities of 8.1–22.2/0.1 mm² and abaxial densities of 11.3–24.5/0.1 mm² in the Pacific Northwest. Total stomatal frequencies are similar in both groups. Stomatal distribution and densities are here shown to not vary significantly within populations and appear to be genetically determined, as shown by progeny tests and growth of seedlings under uniform and experimental conditions. Stomatal distribution in Carex aquatilis appears to be adaptive, and intraspecific variation provides a system for determining the adaptive significance of differences in stomatal patterns.     

FLOATING STOMATES (ADETOSTOMY) IN FERNS: DISTRIBUTION AND ONTOGENY

A unique guard cell apparatus occurs in certain species of Anemia (Schizaeaceae) and in certain species of the unrelated family Polypodiaceae. A guard cell pair is completely surrounded by one epidermal cell, with no attachment to lateral (anticlinal) walls of adjacent epidermal cells. This condition of “floating stomates” is called adetostomy. In rare cases the surrounding epidermal cell is in turn entirely enclosed by another single epidermal cell. The ontogeny of floating stomates has been variously interpreted. This study supports the early view of Rauter that a funnel-shaped guard mother cell is initiated in place entirely within a protodermal cell.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

Total epidermal cell walls of pea stems respond differently to auxin than does the outer epidermal wall alone

The effect of auxin on cell wall mass in the epidermis of third internodes of Pisum sativum L. cv. Alaska grown in dim red light was investigated using epidermal peels, to determine whether epidermal peels reflect the behavior of the outer epidermal cell wall. In contrast to the outer epidermal wall itself, where auxin caused thinning in proportion to growth (M.S. Bret-Harte et al, 1991, Planta 185, 462–471), auxin promoted an increase in wall mass in epidermal peels from treated internode segments in the absence of exogenously supplied sugar. The percentage gain in mass was smaller than the percentage elongation, however, so mass per unit length decreased in peels from auxin-treated segments. Epidermal peels from auxin-treated segments gained more wall mass than control peels even when adhering internal tissue at the basal end of the peel was removed. Epidermal peels also had a gross composition different from that of the outer wall alone (M.S. Bret-Harte and L.D. Talbott, 1993, Planta 190, 369–378). These discrepancies can be explained by the observation that the outer wall makes up only 30% of the mass of the epidermal peel. It appears that the inner walls of the epidermis, and walls of the outer layer of cortical cells that remain attached to the epidermis during peeling, nearly maintain their thickness by biosynthesis while the outer wall loses mass as previously described (Bret-Harte et al. 1991). These results indicate that epidermal peels may not be a good system for examining the biochemical and physiological properties of the outer epidermal cell wall.I would like to thank Dr. Peter M. Ray, of Stanford University, for the use of experimental facilities, helpful discussions, and technical and editorial assistance, Dr. Winslow R. Briggs, of the Carnegie Institute of Washington, for helpful discussions and for the use of experimental facilities, Dr. Paul B. Green, of Stanford University, for financial support, and Dr. Wendy K. Silk, of the Department of Land, Air, and Water Resources, University of California, Davis, for financial support. This work was supported by a National Science Foundation Graduate Fellowship, National Science Foundation grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk in the final writing.     

Anatomical studies of Carex cuchumatanensis,C. decidua,and C. hermannh (Cyperaceae) and comparisons with north American taxa of the C. acuta complex

Foliar anatomy and micromorphology of perigynia and achenes were investigated for three taxa of theCarex acuta complex of sect.Phacocystis Dumort. in Middle and South America. Anatomical characters appear to be conservative, and show little variation amongC. cuchumatanensis Standl. & Steyerm.,C. decidua F. Boott, andC. hermannii Cochrane. Leaves of all species are amphistomatous, with stomates and papillae on both epidermal surfaces, and have a single layer of bulliform cells but differ in the number of vascular bundles. Perigynia have epidermal cells with thin, convex, outer walls that are collapsed in dired specimens, and epidermal cells of achenes each have a single, rounded, nodulose silica-body with a basal platform. Anatomical data are useful in resolving the affinities among these species, and show that they are anatomically indistinguishable fromC. lenticularis Michx. These four New World endemics differe substantially fromC. nigra (L.) Reichard.     

On the laterocytic stomatotype in angiosperms

The laterocytic type of stomatal apparatus in angiosperms is considered. Investigation of the stomatal apparatus of 18 species of Hamamelidaceae, representing 15 genera, showed that in addition to the anomocytic, paracytic, and encyclocytic stomatotypes previously known in the family, the laterocytic type is found in several genera (Dicoryphe, Exbucklandia, and others). Study of 15 species of Chloranthaceae, representing all five genera, showed that laterocytic stomates occur inChloranthus andSarcandra and sometimesHedyosmum, along with stomates of other types.Barbeya oleoides (Barbeyaceae) and the four investigated species ofBalanops (Balanopaceae) have exclusively (or inB. oliviformis mainly) laterocytic stomates. Laterocytic stomates are present also inKadsura andSchisandra of the Schisandraceae, along with the previously known paracytic type. In addition to the foregoing genera, laterocytic stomates are known in some members of the Buxaceae, Celastraceae, Crypteroniaceae, Hydrangeaceae, Icacinaceae Platanaceae, Tetracentraceae and Trochodendraceae.     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Stomatal density and responsiveness of banana fruit stomates

Determination of stomatal densities of the banana peel (Musa acuminata L. var Hort. Valery) by microscopic observations showed 30 times fewer stomates on fruit epidermis than found on the banana leaf. Observations also showed that peel stomates were not laid down in a linear pattern as on the leaf.

It was demonstrated that stomatal responses occurred in banana fruit. Specific conditions of high humidity and light were necessary for stomatal opening: low humidity and darkness were necessary for closure. Responsiveness of the stomates continued for a considerable length of time after the fruit had been severed from the host.

     

Focusing of light by leaf epidermal cells

Leaf epidermal cells from a wide variety of plants focus light to surprisingly high levels. Using image analysis, the concentration and distribution of light was measured after it passed through epidermal cells within peels and epidermal cells attached to palisade cells in partially dissected leaves. In peels taken from Medicago sativa, Zea mays , and Impatiens sp., light was concentrated 15- to 20-fold by individual epidermal cells. When left attached to the mesophyll, which attenuated focusing by absorption and scattering, light was focused up to 5 times. The position of the focal spot beneath each epidermal cell was affected by the direction at which the light struck the cell. When the light was perpendicular to the leaf surface, individual focal spots fell beneath each epidermal cell. When the incident light was oblique, the focal spot shifted laterally and was positioned closer to the anticlinal cell wall. Focusing was observed when leaves were irradiated with collimated light but not with diffuse light. Focal lengths were relatively independent of wavelength within the visible region of the spectrum and there were only slight differences between focusing of blue vs red light. Epidermal lens properties can affect chlorophyll fluorescence and the photosynthetic performance of leaves. A survey of 47 species collected from a wide variety of habitats indicates that many plants have leaf epidermal cells with lens properties. The ability to measure epidermal focusing makes it possible to examine the adaptive and physiological significance of epidermal lens effects in plants.     

Photosystem II in Guard Cells of Vicia faba: Immunological Detection

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.     

Effect of Phenolic Compounds on ABA-induced Changes in K+ Concentration of Guard Cells and in Epidermal Diffusive Resistance

The study of the structure-activity relationship of phenoliccompounds in reversing the ABA-effect on stomata led us to investigatethe changes in K⁺ concentrations in guard cells and in the epidermaldiffusive resistance of leaves, after treatment with ABA andphenolics. The amount of potassium localized in guard cells usually correspondsto stomatal aperture in different treatments. Umbelliferone,however, permits stomatal opening without retention of potassiumin the guard cells, which is an exception. The effect of phenolicsin retaining K⁺ in epidermal peels is matched by recorded epidermaldiffusive resistance changes in the leaves.Although flavonoidsand some other phenolics behave differently showing recoveryin epidermal peels with K⁺ in guard cells, epidermal diffusiveresistance is not recovered. Key words: Epidermal diffusive resistance, K⁺, ABA, phenolics, stomata     

In situ field measurement of leaf water potential using thermocouple psychrometers

Thermocouple psychrometers are the only instruments which can measure the in situ water potential of intact leaves, and which can possibly be used to monitor leaf water potential. Unfortunately, their usefulness is limited by a number of difficulties, among them fluctuating temperatures and temperature gradients within the psychrometer, sealing of the psychrometer chamber to the leaf, shading of the leaf by the psychrometer, and resistance to water vapor diffusion by the cuticle when the stomates are closed. Using Citrus jambhiri, we have tested several psychrometer design and operational modifications and showed that in situ psychrometric measurements compared favorably with simultaneous Scholander pressure chamber measurements on neighboring leaves when the latter were corrected for the osmotic potential.     

Preparation of Cuticle-free Epidermal Peels for the Demonstration of Primary Pit-fields

Cuticle-free epidermal peels for the demonstration of primarypit-fields (if any) on the outer surface of the epidermal cells,can be prepared by a chemically controlled process, using 5-10per cent NaOH solution as a macerating medium. Such peels canbe obtained from any desired part of the plant within a periodof a week. The process involves allowing the trimmed piecesof the plant material to stand in the NaOH solution at 50–60°C, until all the cuticular depositions on the outer surfaceof the epidermal cells are dissolved and the unwanted tissuesare sufficiently loosened to allow the easy removal of the epidermis.The separation of epidermis is done by giving moderate agitationsin water during washing or by means of a soft-haired brush undera dissecting microscope. The peels so obtained must be washed thoroughly in water andcan then be stained with bismark brown or iron alum hematoxylln.After dehydration in ethanol, they can be mounted permanentlyin Canada balsam.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

DIFFUSION THROUGH STOMATES

Ting, Irwin P., and Walter E. Loomis. (Iowa State U., Ames.) Diffusion through stomates. Amer. Jour. Bot. 50(9): 866–872. Illus. 1963.—It is shown that the rule that diffusion through isolated, small pores is proportional to the diameter rather than the area of the pores is valid for pores of diameters as small as 20 μ, and that the curve extends to the origin at zero diameters, indicating that the law is effective throughout the range of stomatal sizes. Suggestions that an elliptical pore will be relatively more effective in diffusion than a circular one and that diffusion is concentrated at the periphery of the pore are not supported by experimental evidence and are physically improbable. Brown and Escombe's conclusion that there is no interference in the diffusion through the individual pores of a multiperforate membrane if the pores are spaced 10 diameters apart is not valid for diffusion through the stomates of a leaf. With pores of 200 μ and less spaced 10 diameters apart, interference increases rapidly with a smaller size and larger number of pores. As a result, the diffusion through a membrane with pores 19 μ in diameter and 190 μ apart was the same as that through a membrane with pores 132 μ in diameter and 1.32 mm apart, although the calculated capacity of the first membrane was 7 times that of the second. The diffusion of water vapor through multiperforate membranes with pores spaced 10 diameters apart has an apparent maximum of 65–70% of the diffusion through an open tube. Calculations of the effect of partial closing of stomates, using Verduin's equation for interference between pores, indicate that the theoretical diffusion capacity of 10 μ stomates spaced at 10 diameters would be increased several times by closing to an average diameter of 5 μ. This increase illustrates the dominant effect of interference in diffusion through small, closely spaced pores. Calculated diffusion through these stomates would not be decreased until they were more than 95% closed. It is concluded that stomatal opening will have no important effect on diffusion from or into a leaf until the stomates are essentially closed.